1. Field of the Invention
The present invention relates generally to the fields of analytical chemistry and biochemistry. More specifically, the present invention relates to direct viral characterization by mass spectrometric detection of capsid proteins.
2. Description of the Related Art
Medical, agricultural, and military personnel who need to monitor and identify microbial agents often utilize enzyme-linked immunosorbent assays, serological, morphological, and other microbiological methods (1). Certain viruses represent a biological threat; therefore, a need exists for rapid detection of characteristic viral biomarkers. Field responses to biological agents require completion of the entire analysis in less than 10 minutes, thereby leaving only minutes for chemical manipulation of the sample.
Since the inception of matrix-assisted laser desorption ionization (MALDI) mass spectrometry (2), the marriage of this desorption/ionization technique with time-of-flight mass spectrometers has enabled scientists to detect large biopolymers with high sensitivity (3). Semi-purified mixtures of proteins have been analyzed with matrix-assisted laser desorption ionization mass spectrometry in the past, thereby providing a more accurate alternative to the commonly used SDS/PAGE analysis of protein mixtures (4). The energy imparted from matrix-assisted laser desorption ionization onto proteins and the conformationally indiscriminate time-of-flight removes some effects of complex protein solution behavior, which can alter the gel migration of different proteins. Mass spectrometry also provides mass measurements in a shorter amount of time than electrophoresis.
Viruses and bacteriophages contain large quantities of proteins, which perform various functions for each virion. One type of protein commonly found in viruses, which is known as a nucleocapsid or coat protein, encapsulates nucleic acid molecules at the core of the organism. In certain more complex virions, this protein capsid layer is surrounded by a lipoglycoprotein layer. The viruses in this study consist of a defined number of homogeneous capsid protein molecules in close association with the viral nucleic acids. FIG. 1 displays the structures of the MS2, tobacco mosaic (TMV) and Venezuelan equine encephalitis (VEE) viruses.
TMV and the MS2 bacteriophage are representative of an RNA virus and bacteriophage respectively. Despite different protein conformations and resulting structural organization, these proteins perform similar functions of acting as translational repressors of the phage-encoded replicase gene, protecting the viral RNA, and serving as a nucleation site for viral assembly. The icosadeltahedron shaped MS2 bacteriophage consist of 180 copies of a coat protein with a molecular weight of 13,730 Da surrounding a single stranded RNA, and one copy of an "A-protein" (MW .about.44 kDa). The TMV virus is a spherical virus consisting solely of coat proteins, which has species dependent molecular weights of 17,100-17,600 Da.
Venezuelan equine encephalitis was used as an example of membrane-containing viruses, which are assembled by interaction of viral proteins with the host cell membrane. Venezuelan equine encephalitis contains a single stranded RNA genome encapsulated by 240 copies of a nucleocapsid protein (M.W. 30,940 Da) surrounded b y a lipid bilayer containing two glycoproteins, E1 and E2 that have molecular masses of .about.50,000 and .about.56,000 Da, respectively (5-7). While the relative abundance of these unique biomarkers presents an opportunity for identification of the virus, scientists have only recently attempted to rapidly identify these biomarkers. Previously, mass spectrometric detection of viral coat proteins required extensive off-line or elaborate on-line cleanup processes. Despeyroux et al. utilized HPLC on-line with electrospray mass spectrometry to determine molecular weights of specific capsid proteins (8).
The prior art is deficient in the lack of effective means of identifying viruses from completely crude biological media with minimal or no cleanup. Further, the prior art is deficient in the lack of effective means for rapid detection of as little as femtomoles of coat protein within a complex biological medium. The present invention fulfills this long-standing need and desire in the art.